CN106916228B - The self assembly series connection cell-penetrating peptide nano particle antibacterial agent and the preparation method and application thereof of blood-brain barrier can be penetrated - Google Patents
The self assembly series connection cell-penetrating peptide nano particle antibacterial agent and the preparation method and application thereof of blood-brain barrier can be penetrated Download PDFInfo
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- CN106916228B CN106916228B CN201710025808.9A CN201710025808A CN106916228B CN 106916228 B CN106916228 B CN 106916228B CN 201710025808 A CN201710025808 A CN 201710025808A CN 106916228 B CN106916228 B CN 106916228B
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- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- NGSWKAQJJWESNS-ZZXKWVIFSA-N trans-4-coumaric acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-N 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses self assembly series connection cell-penetrating peptide nano particle antibacterial agents that can penetrate blood-brain barrier and the preparation method and application thereof.The general structure of the antibacterial agent is Cn- X-Y, wherein X and Y is cell-penetrating peptide, and X and Y connect to form hydrophilic segment；CnIndicate the hydrophobic part fatty acid chain with cell-penetrating peptide coupling；Cell-penetrating peptide is L-type amino acid；The carbon atom number of the fatty acid chain is 12-20.Self assembly series connection cell-penetrating peptide nano particle of the present invention has preferable antibacterium (including drug-resistant bacteria) and antifungic action, it can penetrate blood-brain barrier, and hemolytic toxicity is lower, can be used for developing the novel antibacterial drug for the treatment of brain infection and other infectious diseases.
Description
Technical field
The present invention relates to series connection cell-penetrating peptides, more particularly to the self assembly series connection cell-penetrating peptide nanometer that can penetrate blood-brain barrier
Grain antibacterial agent and the preparation method and application thereof, the application be related to nano particle antibacterial agent preparation treatment brain infection and other carefully
Application in bacterium or fungal infection disease drug.
Background technique
Brain infection is used as one of most important infectious cause of death for a long time, can by bacterium such as staphylococcus aureus,
Escherichia coli and fungi such as Candida albicans cause.In acute cerebral meningitis patient, even if bacterium is sensitive bacteria, and is given very early
Antibiotic treatment is given, high mortality is still likely to occur in a few houres, and survivor can suffer from permanent visual impairment, hearing
Forfeiture, neurological dysfunction and action obstacle.Although the major progress of antibiotic treatment, since drug is difficult to penetrate blood-brain barrier
Into cerebrospinal fluid and brain tissue, brain infection patient still has very high pathogenicity rate and the death rate.It is clinically only a small number of anti-
Raw element can be used for treating brain infection, but the higher toxicity of the organs such as its Central nervous system and liver kidney limits it and makes
With.Another factor for influencing therapeutic effect is the bacterial drug resistance constantly reinforced in recent years and the antibody-resistant bacterium increased.
In recent years, cell-penetrating peptide wears film ability because its is superpower and carries the function vector that molecule enters cell, causes wide
General concern.Poly arginine R9(or polylysine K9) it is famous one of cell-penetrating peptide, and NP1 (or NP2) is Sangho in 2015
The cell-penetrating peptide that Lim etc. has found for the first time from people's NLBP albumen, have it is extremely strong wear film and albumen translocation, and its sequence exists
In in many species bodies.
Self-assembling peptides nano particle is expected to become novel because of the ability of its broad spectrum antibiotic activity and inhibition drug-resistant microorganism
Antibacterials.Therefore, very significant by improving cell-penetrating peptide exploitation novel antibacterial drug.
Summary of the invention
The purpose of the present invention is to provide the self assembly series connection cell-penetrating peptide nano particle antibacterial agent that can penetrate blood-brain barrier, hairs
It is bright to be based on cell-penetrating peptide NP1 (or NP2) and R9(or K9) connect and modified, devise the self assembly that can penetrate blood-brain barrier
Series connection cell-penetrating peptide nano particle antibacterial agent, and it can overcome bacterial drug resistance, can be used for treating brain infection and other infection diseases
Disease.
The purpose of the present invention is achieved through the following technical solutions:
The self assembly series connection cell-penetrating peptide nano particle antibacterial agent of blood-brain barrier can be penetrated: the general structure of the antibacterial agent is
Cn- X-Y, wherein X and Y is cell-penetrating peptide, and X and Y connect to form hydrophilic segment；CnIndicate the hydrophobic part with cell-penetrating peptide coupling
Fatty acid chain；Cell-penetrating peptide is L-type amino acid；The carbon atom number of the fatty acid chain is 12-20；
The X is cationic cell-penetrating peptide；
The Y is NP1 or NP2；The NP1 sequence is KKDKKDERRRK；The NP2 sequence is KIKKVKKKGRK.
To further realize the object of the invention, it is preferable that the fatty acid is lauric acid, myristic acid, palmitinic acid, tristearin
Acid or arachidic acid.
Preferably, the cationic cell-penetrating peptide is nine poly arginine R9Or nine polylysine K9。
Preferably, the self assembly series connection cell-penetrating peptide nano particle antibacterial agent has the average diameter of 100-500nm.
The preparation method of the self assembly series connection cell-penetrating peptide nano particle antibacterial agent for penetrating blood-brain barrier, including it is as follows
Step:
1) using Fmoc solid-phase synthesis preparation series connection cell-penetrating peptide, first swelling resin, after washing and being deprotected, by first
Fmoc- amino acid and HoBt are condensed, and after being deprotected and washing, indenes inspection confirmation deprotection is complete；Then it is condensed second ammonia
Base acid, repeats above step, from the end C- to the end N-, until polypeptide chain synthesis is completed；
2) polypeptide chain that synthesis is completed is cracked with lysate, obtains thick peptide with cold ether precipitating after being filtered under diminished pressure, and lead to
Liquid chromatogram is crossed to be further purified；
3) the fatty acid and end N- of cationic cell-penetrating peptide is coupled in cell-penetrating peptide of connecting, palmitinic acid is added to containing triethylamine and
After being stirred to react in the DMF for cell-penetrating peptide of connecting, leads to nitrogen and remove DMF, cleaned with diethyl ether, and be further purified using dialysis membrane
Crude product.
The self assembly series connection cell-penetrating peptide nano particle antibacterial agent for penetrating blood-brain barrier is preparing anti-golden yellow grape
Application in coccus, Escherichia coli, Methicillin-resistant Staphylococcus aureus or Candida albicans drug.
The self assembly series connection cell-penetrating peptide nano particle antibacterial agent for penetrating blood-brain barrier is golden yellow in preparation treatment brain
Application in color staphy lococcus infection drug.
The preparation method of the self assembly series connection cell-penetrating peptide nano particle antibacterial agent for penetrating blood-brain barrier is will be fatty
Acid is coupled with the end N- of cell-penetrating peptide cationic in cell-penetrating peptide of connecting.CnIt is preferred that when palmitinic acid, self assembly series connection cell-penetrating peptide nanometer
Grain antibacterial agent structure are as follows: C16-R9-NP1、C16-K9-NP1、C16-R9-NP2、C16-K9-NP2.Film is worn in any one self assembly series connection
Peptide nanoparticles antibacterial agent, the average diameter for arriving about 500nm with about 100.
Compared with the existing technology, the invention has the advantages that and the utility model has the advantages that
(1) the present invention provides novel self-assembled nanometer polypeptide, the type of self-assembled nanometer polypeptide is increased.
(2) self assembly of the present invention series connection cell-penetrating peptide nano particle antibacterial agent N-terminal with fatty acid modifying to increase hydrophobicity, just
In the amphipathic of raising peptide, it is easier to the progress of self assembly.It can be self-assembly of in pure water under very low peptide concentration
Nano particle without the mechanisms such as being vigorously stirred, being ultrasonically treated, and has preferable stability.
(3) self assembly of the present invention series connection cell-penetrating peptide nano particle antibacterial agent is to gram-positive bacterium, Gram-negative bacteria,
Fungi and drug-resistant bacteria have preferable bacteriostasis, and its antibacterial effect is much stronger than single cell-penetrating peptide.Therefore, it can be applied to
The novel nano antibacterials for being suitable for clinically using are developed, the treatment of microorganism infection disease is conducive to.
(4) concatenated cell-penetrating peptide, and self assembly are contained in self assembly series connection cell-penetrating peptide nano particle antibacterial agent of the present invention
Series connection cell-penetrating peptide is distributed in nano grain surface afterwards, is conducive to increase permeability of cell membranes, so that self assembly cell-penetrating peptide nanometer
Particle can pass through blood-brain barrier.
(5) self assembly series connection cell-penetrating peptide nano particle antibacterial agent of the present invention can be used for preparing treatment brain infection disease medicine
Object effectively inhibits the microorganism in infected brain tissue.
Detailed description of the invention
Fig. 1 is the three-dimensional molecular structure schematic diagram of 1 gained self assembly of embodiment series connection cell-penetrating peptide.
Fig. 2 is the high-efficient liquid phase chromatogram of 1 gained self assembly of embodiment series connection cell-penetrating peptide, its purity is as the result is shown
95.89%.
Fig. 3 is the Matrix-assisted laser desorption ionization figure of 1 gained self assembly of embodiment series connection cell-penetrating peptide,
Its molecular weight is 2984.6 as the result is shown.
Fig. 4 is the molecular dynamics simulation figure that self assembly series connection cell-penetrating peptide is self-assembled into nano particle in embodiment 3.
Fig. 5 is scanning electron microscope nanotopography figure of the self assembly series connection cell-penetrating peptide in ultra-pure water solution in embodiment 3, wherein
The concentration of peptide sample is 0.5mg/ml.
Fig. 6 is the microorganism shape appearance figure of self assembly series connection cell-penetrating peptide nano particle before and after the processing in embodiment 4.
Fig. 7 be in embodiment 4 self assembly series connection cell-penetrating peptide nano particle to the haemocylolysis figures of human red blood cells.
Fig. 8 is in embodiment 5, is connected after cell-penetrating peptide nano particle by intravenous injection self assembly, cerebrospinal fluid of rats sample
Matrix-assisted laser desorption ionization analysis chart.
Fig. 9 is in embodiment 5, after self assembly series connection cell-penetrating peptide nano particle treatment, in infected brain tissue
Bacteria colony count.
Specific embodiment
In order to better understand the present invention, present invention will be further explained below with reference to the attached drawings and examples, but this hair
Bright claimed range is not limited to the range of embodiment expression.
Embodiment 1
When X is nine poly arginine R9, Y NP2, CnWhen for palmitinic acid long-chain, self assembly is connected cell-penetrating peptide nano particle antibacterial
The general structure of agent are as follows: C16-R-R-R-R-R-R-R-R-R-K-I-K-K-V-K-K-K-G-R-K.Its synthesis mode is as follows:
1, material
Palmitinic acid, Fmoc-Arg (pbf)-OH (N- fluorenes methoxy carbonyl acyl group -2,2,4,6,7- pentamethyl Dihydrobenzofuranes -
5- sulfuryl arginine), Fmoc-Lys (Boc)-OH (N- fluorenes methoxy carbonyl acyl group-N'- tertiary butyloxycarbonyl acyl group-lysine), Fmoc-
Ile-OH (N- fluorenes methoxy carbonyl acyl group-isoleucine), Fmoc-Val-OH (N- fluorenes methoxy carbonyl acyl group-valine), Fmoc-Gly-
OH (N- fluorenes methoxy carbonyl acyl group-glycine), mono- MBHA Resin of Rink Amide, DBLK (hexahydropyridine+DMF), HBTU (O- benzene
And triazol-1-yl-N, N, N, N- tetramethyl urinate hexafluorophosphoric acid rouge) and HOBT (1- hydroxy benzo triazole)；Piperidines, acetic anhydride,
DMF (N, dinethylformamide), TFA (trifluoroacetic acid), NMM (N-methylmorpholine), ether, methanol, DCM (methylene chloride).
2, preparation method
The solid-phase synthesis protected using Fmoc (fluorenes methoxy carbonyl acyl group), processing step are as follows:
(1) the Rink Amide-MBHA Resin (resin) of 20g 0.5mmol/g is weighed in peptide synthesis vessel, is taken
200ml DCM swelling resin 30min, then filters, then takes 300ml DMF washing resin, carries out in three times, each washing
Time is 2min, and 100ml piperidines/DBLK (volume ratio 20%) concussion reaction is added into peptide synthesizer after filtering dry cleaning liquid
30min after reaction, then filters out reaction solution, then washs resin in four times with 400mlDMF, washes after finishing and a small amount of resin is taken to do
Ninhydrin detection test, resin are positive, and following raw material is added into peptide synthesis vessel:
After above-mentioned raw materials add, concussion reaction 30min washs resin with 300ml DMF, often after reaction in four times
Secondary wash time 2 minutes, takes a little resin to do ninhydrin test detection, and resin is negative.
(2) 5ml piperidines/DBLK (volume ratio 20%) concussion reaction 30min is added into peptide synthesis vessel, after reaction
Filter out reaction solution, then wash resin in four times with 40ml DMF, wash finish take a little resin to do ninhydrin test detection, bearing-age tree
Rouge is positive, and following raw material is added into reaction vessels:
After above-mentioned raw materials add, concussion reaction 40min washs resin with 40ml DMF, every time after reaction in four times
Wash time is 2min, and a little resin is taken to do ninhydrin detection, and resin is negative.
(3) (a) raw material in shift step (2) keeps (b) (c) (d) (e) raw material and additional amount constant, repeats step
(2) operation is successively condensed amino acid from the end C- to the end N-.I.e. (a) raw material successively replace with Fmoc-Gly-OH (9.37g),
Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Lys(Boc)-OH(18.74g)、
Fmoc-Val-OH(13.58g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmo-
Ile-OH(14.14g)、Fmoc-Lys(Boc)-OH(18.74g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg
(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg
(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg(pbf)-OH(25.95g)、Fmoc-Arg
(pbf)-OH (25.95g), Fmoc-Arg (pbf)-OH (25.95g), (b) HBTU (15.16g), (c) HOBT (5.40g), (d)
NMM (4.39ml), (e) DMF (160ml) additional amount are constant.
(4) the last one Arg synthesis after, deviate from Fmoc- protecting group, add 5ml piperidines/DBLK (volume ratio 1:
5) 30min is reacted, eluted resin is added 160ml acetic anhydride/DMF (volume ratio 1:2) and reacts 30min, cleans tree with 40ml DMF
Rouge, again with methanol are washed resin 8 times, and DMF is removed.After being dried with nitrogen, with TFA (trifluoroacetic acid)/DCM strong acid lysate (volume
Than 1:1), it cracks 3 hours, the polypeptide that synthesis obtains is cleaved from resin, while sloughing all blocking groups, collected molten
There is the lysate of synthetic peptide, be then filtered under diminished pressure, collect filtrate, the polypeptide being dissolved in filtrate is precipitated with cold ether, then filter
White solid is obtained to get the peptide crude product of series connection cell-penetrating peptide.Peptide crude product collects main peak, through being subcooled through high-efficient liquid phase chromatogram purification
Be lyophilized it is dry after, by palmitinic acid (51.23g) and cationic cell-penetrating peptide R9The end arginine N- coupling obtain C16R9NP2, step is such as
Under: the palmitinic acid for being dissolved in 15mL DMF is added slowly to 5mL under 0 DEG C of stirring and contains 70 μ L triethylamines and cell-penetrating peptide of connecting
In DMF.After 24 hours of reacting, DMF is removed from mixture by purging drying nitrogen, then uses diethyl ether washed mixture 3
It is secondary to remove unreacted palmitinic acid, i.e., completion N-terminal modification.It is dialysed 6 days using the film DMF that molecular weight cutoff is 1000Da
Crude product is further purified.Then final product C is obtained to generate final product by vacuum drying removal DMF16R9NP2.Pass through
HPLC and MALDI-TOF analytical proof C16R9The successful synthesis (as follows) of NP2.
3, product detection
Synthesize obtained C16R9For NP2 two-dimensional molecular structure by ChemBioDraw Software on Drawing, three-dimensional separation flow is logical
Cross ChemBio 3D Software on Drawing.As shown in Figure 1, passing through the spatial distribution of its amino acid known to the figure and fatty acid chain.In figure
As it can be seen that the cationic end cell-penetrating peptide N- and palmitinic acid are coupled in series connection cell-penetrating peptide, NP2 is end structure.
The C obtained by hplc rp-hplc detection synthesis16R9NP2 purity.With Cromasil-C-18 column (5
μm) it is used as stationary phase, the mixture of acetonitrile and water records the peak shape of 0-30min as mobile phase.Testing result is shown in Fig. 2, in figure
Corresponding peak is target product peak at 14.032min, determines that its purity reaches 95.89% according to peak area percent result.
By isometric peptide solution (1.0mg/mL) and alpha-cyano -4- hydroxycinnamic acid (HCCA) solution, (saturation is in volume
Than the acetonitrile solution for 50%) mixing, is detected by Matrix-assisted laser desorption ionization MALDI-TOF and is closed
At obtained C16R9NP2.Testing result is shown in Fig. 3, maximum intensity peak in figure, that is, corresponds to point that the peak that numerical value is 2584.6 is product
Daughter ion peak indicates that its molecular weight is 2584.6, consistent with the theoretical molecular weight calculated by structural formula, as a result demonstrates target production
Object C16R9The synthesis of NP2.
Embodiment 2
The self assembly series connection cell-penetrating peptide nano particle for penetrating blood-brain barrier of other fatty acid modifyings of the present invention is anti-
Microbial inoculum can be closed by the amino acid and fatty acid in alternative embodiment 1 using the solid phase of Fmoc (fluorenes methoxy carbonyl acyl group) protection
Cheng Fa is obtained according to preparation method synthesizing and purifying described in embodiment 1.
I.e. by described in the preparation method (1) of embodiment 1, then swellable resins are washed with DMF, and piperidines/DBLK shake is added
Resin is washed after swinging reaction, amino acid starting material, HBTU, HOBT, NMM, DMF are added after ninhydrin tests positive, concussion is anti-
Resin is washed with DMF after answering, ninhydrin inspection is negative.Then described in the preparation method (2) according to embodiment 1, root
According to the amino acid sequence of different series connection cell-penetrating peptides, different aminoacids raw material (sequentially from the end C- to the end N-) is sequentially added, repeats to shake
Swing reaction, washing, ninhydrin detecting step.Again by described in the preparation method (3) of embodiment 1, through deprotection, DMF washing, split
Liquid cracking, ether precipitating, purifying are solved, the synthesis of series connection cell-penetrating peptide is completed.Finally according to institute in the preparation method (4) of embodiment 1
It states, fatty acid is added in triethylamine and the DMF solution for cell-penetrating peptide of connecting and is reacted, complete N-terminal through nitrogen purging, diethyl ether cleaning
Modification.It is purified by dialysis membrane, obtains the series connection cell-penetrating peptide of the i.e. different fatty acid modifyings of final product after dry removal DMF.
Reversed-phase high performance liquid chromatography detection is carried out by detection method in embodiment 1 again to fly with substance assistant laser desorpted ionized
The product that row time mass spectrum detects.When the preferred palmitinic acid of fatty acid, it is as follows to obtain sequence: C16-R9-NP1、C16-K9-NP1、
C16-R9-NP2、C16-K9-NP2.Different compound experiment parameters and testing result are as shown in table 1 below in the process:
The different compound experiment parameters and inspection of the portion self-assembles of the present invention of table 1 series connection cell-penetrating peptide nano particle antibacterial agent
Survey result
Embodiment 3
Self assembly series connection cell-penetrating peptide nano particle antibacterial agent C16R9The self assembly molecule dynamics simulation and scanning electron microscope of NP2
Morphology observation
1, molecular dynamics simulation
To self assembly series connection cell-penetrating peptide nano particle antibacterial agent C in embodiment 116R9NP2 carries out molecular dynamics simulation.Institute
It is Material Studio with software, by Coarse grained model, the Flory-Huggins parameter of itself and water effect is calculated, into one
Step establishes the field of force, carries out molecular dynamics simulation.Analog result is as shown in figure 4, cell-penetrating peptide self assembly shape in aqueous solution of connecting
At nano particle, wherein internal is hydrophobicity palmitinic acid, outside is series connection cell-penetrating peptide.
2, scanning electron microscope morphology observation
C is observed using cold field emission scanning electron microscope (JSM-6330F)16R9The self assembly form of NP2 in aqueous solution.With super
Pure water is by C16R9NP2 is configured to 0.5mg/mL peptide solution, takes 20 μ L peptide solutions to be placed on mica sheet, and natural wind at room temperature
It is dry.Mica sheet is fixed on aluminium column, it is then gold-plated for observing.Fig. 5 is 14000 times of lower acceleration voltages when being 15.0keV
C16R9The scanning electron microscope (SEM) photograph of NP2 aqueous solution, the results showed that the series connection cell-penetrating peptide C of palmitinic acid modification16R9NP2 self assembly shape in water
It is less than the particle of 150nm at diameter.
The embodiment self assembly connect cell-penetrating peptide nano particle antibacterial agent N-terminal with fatty acid modifying to increase hydrophobicity, just
In the amphipathic of raising peptide, it is easier to the progress of self assembly.It can be self-assembly of in pure water under very low peptide concentration
Nano particle without the mechanisms such as being vigorously stirred, being ultrasonically treated, and has preferable stability.
Embodiment 4
Self assembly series connection cell-penetrating peptide nano particle antibacterial agent C16R9NP2 is preparing the application in novel nano antibacterials
1, minimal inhibitory concentration
Strain subject is all from Guangdong microorganism germ plasma resources bank.Staphylococcus aureus ATCC 29213, Escherichia coli
CMCC 44825, methicillin-resistant-staphylococcus aureus ATCC 43300 are trained in 37 DEG C of nutrient agar
It supports.Candida albicans ATCC 10231 is cultivated in 37 DEG C of potato dextrose medium.Using micro-dilution method come
Measure the minimal inhibitory concentration MIC of peptide nanoparticles.In simple terms, the self assembly that 50 μ l concentration are 4.2 to 100.4 μM is connected
Cell-penetrating peptide nanoparticles solution and reference substance R9Peptide solution is placed in each hole of 96 orifice plates, then by the tested bacterium solution of same volume
It is added in each hole to obtain at 600nm 0.1 to 0.2 optical density readings.It is placed in 37 DEG C of shaken cultivation case and incubates
10~16h is dense with the minimum drug for not observing bacterial growth every 2 hours OD values at microplate reader measurement 600nm
Degree is the minimum inhibitory concentration of the sample.Test is in triplicate.
Experimental result, self assembly series connection cell-penetrating peptide C16R9NP2 nano particle and positive control cell-penetrating peptide R9Minimum suppression
Bacteria concentration measurement result is as shown in table 2.
2 lowest bacteria fogging-resistant concentration determining result of table
The result shows that self assembly series connection cell-penetrating peptide nano particle of the present invention all has preferably test microbes
Fungistatic effect, the minimal inhibitory concentration to staphylococcus aureus is 13.4 μM, to Escherichia coli, Methicillin-resistant Staphylococcus aureus
Minimal inhibitory concentration is 8.4 μM, and the minimal inhibitory concentration to Candida albicans is 50.3 μM.And the single cell-penetrating peptide of non-self assembly
R9It is extremely low to the rejection ability of test bacterium and fungi, show as high minimal inhibitory concentration.The formation of nano particle is obvious
The antibacterial ability of peptide is enhanced, minimal inhibitory concentration, therefore self assembly of the present invention series connection cell-penetrating peptide nano particle are reduced
The effective antibacterials for inhibiting microbial growth can be developed into.
2, scanning electron microscope observation microorganism pattern variation
By cultured bacterium solution and be concatenated cell-penetrating peptide nano particle processing bacterium solution, 6000rpm be centrifuged 10min
Afterwards, cell is washed three times with phosphate buffered saline solution (PBS), the then fixation overnight in the PBS containing 5% formaldehyde.With a system
Column concentration gradient ethyl alcohol is dehydrated, and is then freeze-dried, and is fixed on aluminium column.With gold coating sample before sem analysis.Fig. 6
Middle a is the staphylococcus aureus of blank control, and eucaryotic cell structure is full complete, and cell surface is in smooth circle；B is to be concatenated
Significant change, observable occur for cell-penetrating peptide nano particle (minimal inhibitory concentration) treated staphylococcus aureus, pattern
To apparent cell rupture and cell fragment；C is the Candida albicans of blank control, and cell surface is completely smooth；D is through going here and there
Join cell-penetrating peptide nano particle (minimal inhibitory concentration) Candida albicans that treated, significant change occurs for pattern, it is seen that thin
After birth disappears molten, and cell cracking is dead.The microorganism pattern variation that scanning electron microscope is observed demonstrates present invention series connection and wears
Film peptide nanoparticles C16R9The antibacterial ability of NP2 shows that its mechanism of action predominantly destroys microbial cell structure.
3, haemocylolysis detects
Fresh human red blood cells are washed with PBS, and are diluted to 4% (by volume) suspension, and 100 μ L red cell suspensions is taken to set
In each hole of 96 orifice plates, 100 μ L peptide nanoparticles or amphotericin B solution (water solubility) then are added to every hole, 37
DEG C incubate 1 hour.Cell suspension is taken out later and is transferred to 96 orifice plates in 1000g centrifuging and taking equal portions supernatant (100 μ L), utilizes enzyme mark
Instrument is in 540nm monitoring hemoglobin release.
Haemocylolysis percentage is calculated using following equation:
Haemocylolysis (the %)=[(O.D. in nanoparticles solution540nmO.D. in-PBS540nm)/(0.1%
O.D. in TritonX-100540nmO.D. in-PBS540nm)]x100
As a result as shown in fig. 7, nano particle has Low haemolysis effect, in 25mg/L, to Escherichia coli and methicillin-resistant
The minimal inhibitory concentration of S. aureus L-forms, haemocylolysis is less than 4%；It is antibacterial dense to the minimum of staphylococcus aureus in 50mg/L
Degree, haemocylolysis is less than 5%.Under comparable sodium, cell-penetrating peptide nano particle of connecting is than positive control medicine anphotericin (water
Dissolubility) haemocylolysis it is low.Clinically think the medical material less than 5% haemocylolysis be it is safe, therefore, self assembly series connection
Cell-penetrating peptide nano particle C16R9The Low haemolysis effect of NP2 shows its safety as effective antibacterials, can be used to feel
Contaminate the treatment of disease.
Embodiment 5
Self assembly series connection cell-penetrating peptide nano particle antibacterial agent C16R9NP2 penetrates the detection of blood-brain barrier, and in treatment brain
Application in portion's infection
The program of related to animal obtains Institute of Experimental Animals, Chinese Academy of Medical Sciences's experimental animal use and pipe
The approval of the reason committee (IACUC), and according to National Institute of Health Guide for the Care
Refer to described in and Use of Laboratory Anima1s (NIH Publications NO.85-23, revise for 1996)
Southing row.
1, the blood-brain barrier of wearing of self assembly series connection cell-penetrating peptide nano particle acts on
Experimentation passes through tail with reference to Changhong etc. (new method that cerebrospinal fluid of rats extracts, Laboratory Animal Science, 2012) is appointed
Vein injects C to adult SD rats (weight is about 250g)16R9The pure nanoparticles solution of NP2.Take cerebrospinal fluid, 1000g centrifuging and taking
Supernatant is detected by Matrix-assisted laser desorption ionization.Spinal fluid samples testing result is as shown in figure 8, divide
The corresponding numerical value in daughter ion peak is 2984.254, with C16R9NP2 marks product 2984.56 unanimously, therefore is the characteristic peak of peptide nanoparticles,
The result shows that peptide nanoparticles can pass through blood brain barrier of rats, into cerebrospinal fluid, and then brain tissue can be traveled further into.
2, effect of the self assembly series connection cell-penetrating peptide nano particle in treatment brain bacterium infection
Experimentation is with reference to (Self-assembled cationic peptide such as Yi-Yan Yang
Nanoparticles as an efficient antimicrobial agent, Nature nanotechnology,
2009).The staphylococcus aureus liquid that 50 μ L are injected to SD In The Medulla Oblongata of The Rat pond injects C by the every 12h of tail vein16R9NP2 nanometers
Particle solution, blank control group injecting normal saline, positive controls inject vancomycin solution.48h Hou Qu rat cerebral tissue
It weighs and is homogenized, take 100 μ L homogenates to be coated on nutrient agar, calculate its clump count, i.e. Colony Forming Unit, with
Lg Colony Forming Unit/Borneo camphor tissue indicates.As a result as shown in figure 9, after peptide nanoparticles are treated, rat cerebral tissue's clump count
Blank control group (Ji Wei treatment group) is compared to significantly reduce, it is close with through anphotericin treatment group result, show that it is big in treatment
Positive effect is achieved in the infection of mouse brain S. aureus L-forms, it is suppressed that bacterium increases.Therefore, film is worn in self assembly series connection of the present invention
Peptide nanoparticles have a good application prospect in the antibacterials of preparation treatment brain infection disease, can be effectively suppressed and are felt
The growth of the brain bacterium of dye.
Embodiment of the present invention are not limited by the above embodiments, other any real without departing from spirit of the invention
Made changes, modifications, substitutions, combinations, simplifications under matter and principle, should be equivalent substitute mode, are included in the present invention
Protection scope within.
Claims (4)
1. the self assembly series connection cell-penetrating peptide nano particle antibacterial agent of blood-brain barrier can be penetrated, which is characterized in that the knot of the antibacterial agent
Structure general formula is Cn- X-Y, wherein X and Y is cell-penetrating peptide, and X and Y connect to form hydrophilic segment；CnIt indicates and cell-penetrating peptide coupling
Hydrophobic part fatty acid chain；Cell-penetrating peptide is L-type amino acid；The carbon atom number of the fatty acid chain is 12-20；
The X is nine poly arginine R of cationic cell-penetrating peptide9；
The Y is NP2；The NP2 sequence is KIKKVKKKGRK；
The self assembly series connection cell-penetrating peptide nano particle antibacterial agent has the average diameter of 100-500nm.
The cell-penetrating peptide nano particle antibacterial agent 2. self assembly described in claim 1 is connected, which is characterized in that the fatty acid is the moon
Cinnamic acid, myristic acid, palmitinic acid, stearic acid or arachidic acid.
3. the preparation method of the self assembly series connection cell-penetrating peptide nano particle antibacterial agent of blood-brain barrier can be penetrated described in claim 1,
It is characterized by comprising following steps:
1) using Fmoc solid-phase synthesis preparation series connection cell-penetrating peptide: first swelling resin, after washing and being deprotected, by first
Fmoc- amino acid is condensed under HoBt catalysis with resin, has been reacted after being deprotected and being washed, indenes inspection confirmation deprotection
Completely；Then it is condensed second amino acid, repeats above step, from the end C- to the end N-, until polypeptide chain synthesis is completed；
2) polypeptide chain that synthesis is completed is cracked with lysate, obtains thick peptide with cold ether precipitating after being filtered under diminished pressure, and pass through liquid
Phase chromatography is further purified；
3) fatty acid is coupled with the end N- of cell-penetrating peptide cationic in cell-penetrating peptide of connecting: palmitinic acid is added to containing triethylamine and series connection
After being stirred to react in the DMF of cell-penetrating peptide, leads to nitrogen and remove DMF, cleaned with diethyl ether, and thick production is further purified using dialysis membrane
Object.
4. the self assembly series connection cell-penetrating peptide nano particle antibacterial agent that can penetrate blood-brain barrier described in claim 1 is preparing anti-white
Application in candida albicans drug.
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| PCT/CN2017/110896 WO2018130000A1 (en) | 2017-01-13 | 2017-11-14 | Self-assembled serially-connected cell-penetrating peptide nano-particulate antibacterial agent capable of penetrating blood brain barrier, preparation method therefor, and use thereof |
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| CN110123658B (en) * | 2019-05-22 | 2022-07-15 | 上海璞萃生物科技有限公司 | Supermolecule polypeptide with self-assembly aggregate structure and preparation method thereof |
| CN113135984B (en) * | 2021-05-06 | 2022-08-09 | 中国医学科学院放射医学研究所 | In-situ self-assembly polypeptide derivative responding to pathological microenvironment and application thereof |
| WO2023277628A1 (en) * | 2021-07-02 | 2023-01-05 | 주식회사 아임뉴런 | Novel cell-penetrating peptide and use thereof |
| CN114699387B (en) * | 2022-03-17 | 2023-05-23 | 常州大学 | Drug-loaded nanoparticle with core-shell structure and preparation method and application thereof |
| CN115607677A (en) * | 2022-05-06 | 2023-01-17 | 太阳雨林(北京)生物医药有限公司 | Compound for preventing, preventing or treating microbial infection and preparation and application thereof |
| CN114788872A (en) * | 2022-05-06 | 2022-07-26 | 太阳雨林(北京)生物医药有限公司 | Compound for preventing, preventing or treating microbial infection and preparation and application thereof |
| CN115536730B (en) * | 2022-09-20 | 2023-08-15 | 南开大学 | Polypeptide crossing blood brain barrier and preparation method thereof, nanostructure and preparation method and application thereof |
| CN116808231B (en) * | 2023-07-07 | 2024-03-26 | 中国药科大学 | Cell membrane penetrating peptide coupled sulpride prodrug system, preparation method and application |
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| CN103467579A (en) * | 2013-08-26 | 2013-12-25 | 华南理工大学 | Cation amphipathic self-assembly nano antibacterial peptide and application thereof |
| KR20140046996A (en) * | 2012-10-09 | 2014-04-21 | 한양대학교 산학협력단 | Cell penetrating peptide comprising np12 polypeptide or np21 polypeptide derived from human nlbp and cargo delivery system using the same |
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| KR20140046996A (en) * | 2012-10-09 | 2014-04-21 | 한양대학교 산학협력단 | Cell penetrating peptide comprising np12 polypeptide or np21 polypeptide derived from human nlbp and cargo delivery system using the same |
| CN103467579A (en) * | 2013-08-26 | 2013-12-25 | 华南理工大学 | Cation amphipathic self-assembly nano antibacterial peptide and application thereof |
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| 小分子肽自组装行为研究及其生物医学应用;许小丁;《中国博士学位论文全文数据库（工程科技Ⅰ辑）》;20150415(第04期);参见第11页第10行至第16页第1行，图9 |
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